Efforts to develop a protective HIV-1 vaccine have been hindered by difficulties in identifying epitopes capable of inducing broad neutralizing antibody responses. In fact, the high mutation rate occurring in the gene encoding HIV-1 envelope proteins and the complex structure of gp120 as an oligomer associated with gp41 result in a high degree of antigenic polymorphism. Furthermore, a vaccine formulation should take into account the subtype prevalence in different geographic regions as well as the insurgence of new HIV isolates. To overcome these obstacles, we screened Random Peptide Libraries (RPL) using sera from HIV-infected subjects in order to identify antigenic and immunogenic mimics of HIV-1 epitopes. After extensive counter-screening with HIV-negative sera, we isolated peptides specifically recognized by antibodies from HIV-1 infected individuals. These peptides behaved as antigenic mimics of linear or conformational HIV-1 or SHIV epitopes generated in vivo in the course of natural infection. The selected epitopes were immunogenic in mice, where they elicited HIV-specific antibodies that effectively neutralized HIV-1 isolates. Although the HIV-1 mimotopes were isolated by using clade B sera, they reacted with antibodies from subjects infected with different HIV-1 clades, including clade A, C, D, E, and F, suggesting that they function as antigenic mimics of HIV-1 epitopes shared by multiple HIV-1 strains and subtypes from distant geographic regions. Consistent with these findings, sera of monkeys infected with SHIVs carrying envelopes from different primary isolates, such as DH12, 89.6 and 89.6P, also recognized the pool of HIV-specific epitopes. When injected in Rhesus macaques with QS21 adjuvant, a pool of five epitopes induced an antibody response specific for each of the single epitope and this response cross-reacted with HIV-1 envelope proteins. These animals have been recently challenged intravenously with the highly pathogenic strain SIHV89.6PD to evaluate the level of protection induced by the selected epitopes. The study should assist in developing an effective HIV-1 vaccine.